In humans, coronaviruses are one of the viral agents causing the common cold and are implicated as the causative agent of necrotizing enterocolitis in newborns and of gastroenteritis in children and adults. In domesticated animals, coronaviruses produce a broad range of diseases including gastroenteritis, encephalitis, and respiratory disease. The ability of coronaviruses to cause persistent infection and produce a carrier state in otherwise healthy individuals contribute to their enigma. The long range goal of our research effort is to understand the underlying replication strategy of viruses belonging to the Coronaviridae and to relate this replication strategy to their ability to establish and maintain a persistent infection and to produce disease. The ability of coronaviruses to replicate depends on the successful synthesis of negative strand molecules which in turn serve as the templates for the synthesis of viral genomes and subgenomic messenger RNA. We were able extend the recent report of subgenomic negative strands in cells infected with transmissible gastroenteritis virus of swine. We found that mouse hepatitis virus infected cells also produced subgenomic-sized replication intermediates which suggest that probably all coronaviruses possess a unique and as yet undefined replication strategy. The immediate goal of this research proposal is to define the mechanism by which coronaviruses produce subgenomic negative strands. We propose to use temperature sensitive mutants to characterize coronavirus negative-strand RNA synthesis and to evaluate the synthesis of negative strands in both lytically and persistently infected cells.